Use of s-nitrosothiol signaling to treat disordered control of breathing

ABSTRACT

The present invention is directed to a method of treating disordered control of breathing including the treatment of apnea and hypoventilation associated with congenital or acquired brain stem abnormalities. Specifically the invention is directed to treating disordered control of breathing by administering an S-nitrosylating agent selected from the group consisting of ethyl nitrite, glutathione, nitric oxide, S-nitrosocysteine, S-nitrosoglutathione, S-nitro-N-acetyl cysteine. As shown in FIG.  1 C the ability of endogenous SNO g  to increase V E  in freely behaving, conscious rates using whole-body plethysmography revealed that CSNO, GSNO and CGSNO (1 nmol each) caused equivalent increases in V E , whereas D-CSNO had no effect (left bar graph is the equivalent increases in V E , whereas D-CSNO had no effect (left bar graph is the control whereas the right bar represents administration of the respective SNO).

RELATED APPLICATION

[0001] This application claims priority under 35 USC §199(e) to U.S.Provisional Application Ser. No. 60/313,548, filed Aug. 20, 2001, thedisclosure of which is incorporated herein.

US GOVERNMENT RIGHTS

[0002] This invention was made with United States Government supportunder Grant Nos. HL 59337, awarded by the National Institutes of Health.The United States Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention is directed to compositions and methods fortreating disordered control of breathing. More particularly, the presentinvention is directed to the use of S-nitrosothiols or otherS-nitrosylating agents to enhance ventilation in patients suffering froma congenital or acquired brain stem abnormality.

BACKGROUND OF THE INVENTION

[0004] The ability to increase minute ventilation (V_(E)=respiratoryrate times tidal volume) in response to hypoxia is essential forsurvival. Failure to breathe more often when oxygen levels are low cancontribute to respiratory distress in newborn mammals and to sleep apneain adults. The mechanisms by which hypoxic stimuli are processed arepoorly understood. However, it is known that V_(E) increases linearlywith decreasing oxyhaemoglobin saturation (about 0.6 l/min percentsaturation in healthy individuals) and that its regulation involvesinput to brainstem areas such as the NTS, that are rich in nitric oxidesynthase (NOS). This increase in breathing is regulated not only by themere lack of oxygen, but rather by molecules related to a different gas,nitric oxide (NO), which affect respiratory centers at the base of thebrain. These respiratory centers include neurons present in the brainstem as well as those in the carotid body.

[0005] Abnormalities of central control of ventilation, particularly inresponse to hypoxia, can be life-threatening. Central apnea andhypoventilation occur in patients with congenital and acquired brainstem abnormalities, ranging from Arnold Chiari malformation to scartissue associated with treatment of brainstem tumors. Furthermore, anapneic or hypoventilatory response to hypoxemia can occur in patientswith obstructive sleep apnea, and abnormal dependence on hypoxicventilatory drive can also make oxygen therapy life-threatening inpatients with chronic obstructive lung diseases. A newborn infant(particularly the premature and/or anemic infant) can have a paradoxicalapneic or hypoventilatory response to hypoxemia that is believed to playa role in the pathogenesis of some cases of Sudden Infant DeathSyndrome. Other patients may have profound paradoxical hypoventilationwhen asleep, as seen in congenital central hypoventilation syndrome.

[0006] Current therapeutic options for each of these disorders arelimited primarily to techniques involving artificial ventilation. Ofnote, therapeutic options for respiratory alkalosis associated withacute hyperventilation (whether psychiatric or drug-induced) aresimilarly limited. The present invention is directed to a novel approachto the treatment of disorders of control of breathing that is based onthe use of nitrosylating agents to enhance minute ventilation in suchindividuals.

SUMMARY OF THE INVENTION

[0007] The present invention is directed to a novel approach to treatingapnea and hypoventilation associated with congenital or acquired brainstem abnormalities. In particular, applicants have demonstrated that aclass of endogenous compounds known as S-nitrosothiols dramaticallyincreases minute ventilation (V_(E)) at the level of the brainstemrespiratory control centers in the nucleus tractus solitarius (nTS). Inaccordance with one embodiment a composition comprising low molecularweight reduced thiols or an S-nitrosylating agent is provided fortreating disordered control of breathing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1A-1C Ventilatory effects of SNOs in rats. FIG. 1A is a graphplotting V_(E) vs Time and representing the V_(E) during (shaded) andfollowing a short period of hypoxia. FIG. 1B is a graph plotting V_(E)vs Time and representing the results obtained after injecting 1 nmolS-Nitrosocysteinyl glycine (CGSNO) into the nucleus tractus solitarius(NTS). A marked increase in V_(E) (injection indicated by an arrow) wasobserved with onset and decay characteristics identical to thoseobserved during short exposure of the whole animal to hypoxia and returnto normoxia. FIG. 1C is a bar graph representing the data obtained afteradministering various L-SNO isomers. All L-SNO isomers caused increasesin V_(E) (change from baseline for CGSNO: asterisk, P<0.001, n=10;S-nitrosoglutathione (GSNO): asterisk, P<0.0001, n=14;S-nitroso-L-cysteine (L-CSNO): asterisk, P<0.0001, n=20), whereasS-nitroso-D-cysteine (D-CSNO) was without effect (P=NS; n=20)

[0009]FIGS. 2A & 2B are bar graphs demonstrating the effect ofγ-glutamyl transpeptidase (GGT) inhibition or deficiency on theventilatory effects of SNOs. FIG. 2A shows that the V_(E) increasesstimulated by microinjection of 10 nmol GSNO were abolished afterpre-treatment with the GGT inhibitor acivicin (7.5 nmol; P<0.0001; n=8).CGSNO (10 nmol) stimulated V_(E) increases that were not modified byacivicin (P=NS; n=6). FIG. 2B represents the data generated from ahypoxic ventilatory response in GGT-deficient mice (+/+, wild type; +/−,heterozygotes; −/−, GGT-deficient). Lowest V_(E) during the 30 s aftercessation of hypoxia is expressed as percent change from pre-hypoxiabaseline (P<0.0001; n=8 each group).

DETAILED DESCRIPTION OF THE INVENTION

[0010] Definitions

[0011] In describing and claiming the invention, the followingterminology will be used in accordance with the definitions set forthbelow.

[0012] As used herein, the term “treating” includes alleviating thesymptoms associated with a specific disorder or condition and/orpreventing or eliminating said symptoms.

[0013] As used herein, an “effective amount” means an amount sufficientto produce a selected effect. For example, an effective amount of aS-nitrosylating agent for treating disordered control of breathing is anamount sufficient to alleviating the symptoms associated with disorderedcontrol of breathing, including enhancing minute ventilation (V_(E)).

[0014] As used herein the term “disordered control of breathing” relatesto any disease state or condition that causes a neurological-based lossof the ability to regulate respiration in the afflicted individual.Disorders that relate to the control of breathing include apnea andhypoventilation that is associated with congenital or acquired brainstem abnormalities in addition to neurological abnormalities of nervesinnervating respiratory muscles, and nerves signaling from peripheralchemoreceptor. Note that lung disorders can lead to secondary disordersof control of breathing, such as blunted hypoxic ventilatory driveobserved in chronic obstructive pulmonary disease.

[0015] As used herein the term “nitrosylation” refers to the addition ofNO to a thiol group (SH), oxygen, carbon or nitrogen by chemical means.An “S-nitrosylating agent” refers to a compound that can function invivo to react with protein thiol groups, transferring a NO group to thethiol to form an S-nitrosothiol. Suitable nitrosylating agents aredisclosed in Feelisch and Stamler, “Donors of Nitrogen Oxides”, Methodsin Nitric Oxide Research edited by Feelisch and Stamler, (John Wiley &Sons) (1996), the entire teachings of which are hereby incorporated intothis application by reference. S-nitrosylating agents include acidicnitrite, nitrosyl chloride, ethyl nitrite, glutathione,S-nitrosoglutathione, S-nitrosocysteinyl glycine, S-nitrosocysteine,N-acetyl cysteine, S-nitroso-N-acetyl cysteine, nitroglycerine,nitroprusside, nitric oxide, S-nitrosohemoglobin and S-nitrosoalbumin.

[0016] As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially free(i.e. at least 60% free, preferably 80% free, and most preferablygreater than 90% free) from other components with which they arenaturally associated.

[0017] As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water and emulsions such as anoil/water or water/oil emulsion, ethanol and various types of wettingagents.

[0018] As used herein, the term “parenteral” includes administrationsubcutaneously, intravenously or intramuscularly.

[0019] The Invention

[0020] Applicants have discovered that S-nitrosothiols, includingS-nitrosoglutathione (GSNO) dramatically increase minute ventilation(V_(E)) at the level of the brainstem respiratory control centers in thenucleus tractus solitarius (nTS), see FIG. 1. Furthermore, GSNO may beformed during blood deoxygenation and is present in μM concentrations inthe mammalian brain stem. Applicants have also shown (see FIG. 2) thatthe GSNO-induced increase in V_(E) is dependent on the presence andactivity of an enzyme, γ-glutamyl transpeptidase (GGT). GGT breaks downGSNO to S-nitrosocysteinyl glycine (CGSNO). When GGT is inhibited,CGSNO, but not GSNO, increases minute ventilation. Importantly, GSNO hasno effects on blood pressure or heart rate (“hemodynamic effects”) inthe same species. Therefore, GGT-dependent GSNO effects 1) stimulateincreased ventilation; 2) translate the effect of hypoxia to signal arespiratory effect at the level of the brain stem; 3) separatesrespiratory from hemodynamic responses at the level of the nTS; and 4)are regulated by GGT.

[0021] This pathway can be exploited through the use of modifiers ofS-nitrosoglutathione metabolism to treat disorders of the control ofbreathing. For example, such disorders can be treated by theadministration of GSH or GSH-mimetics, by administration of additionalS-nitrosothiol precursors, or by stimulation of GGT. In accordance withone embodiment the method comprises the step of administering to aindividual suffering from a breathing disorder a compound selected fromthe group consisting of ethyl nitrite, glutathione,S-nitrosoglutathione, S-nitrosocysteinyl glycine, S-nitrosocysteine,N-acetyl cysteine, S-nitroso-N-acetyl cysteine and nitric oxide. In onepreferred embodiment the nitrosylating agent is selected from the groupconsisting of N-acetyl cysteine, S-nitroso-N-acetyl cysteine, ethylnitrite, glutathione and S-nitrosoglutathione.

[0022] N-acetyl cysteine, ethyl nitrite, nitric oxide, N-acetyl cysteineand glutathione represent S-nitrosothiol precursors that can be modifiedin vivo to become agents capable of nitrosylating proteins. Compositionscomprising nitrosylating agents or S-nitrosothiol precursor compoundscan be further supplemented with agents that stimulate GGT activity. Inone alternative embodiment the composition used for treating disorderedcontrol of breathing comprises a stimulant of γ-glutamyl transpeptidaseactivity as the active agent. Compounds that stimulate GGT activityinclude retinoic acid and other retinols and stimulants of retinoic acidreceptors, follicle stimulating hormone, ethoxyquin and other stimulantsof GGT promoter III or inhibitors of GGT gene negative regulatory sites,glycine-glycine in conjunction with glutathione, prostaglandin E2 withdiethylnitrosamine, and stimulants of the antioxidant response elementpromoter. The disorders of control of breathing that can be treated inaccordance with the present invention include central apnea, centralhypoventilation, impaired control or peripheral respiratory drive,respiratory fatigue complicating obstructive lung disease, obstructivesleep apnea, and impending respiratory failure.

[0023] The S-nitrosylating agents of the present invention can beformulated with pharmaceutically acceptable carriers, diluents, andsolubilizing agents for administration to a patient in need of suchtherapy. Such administration can be, for example, by injection (in asuitable carrier, e.g., sterile saline or water), inhalation, oral,transdermal, rectal, vaginal, or other common route of administration.The route of administration selected will vary based on the condition tobe treated and the S-nitrosylating agent used to treat the individual.For example, N-acetyl cysteine, S-nitroso-N-acetyl cysteine andS-nitrosoglutathione are suitable for oral or inhalation administration.Whereas NO is only suitable for inhalation.

[0024] In one embodiment an S-nitrosylating agent, such asS-Nitrosoglutathione or other S-nitrosothiols, is administeredintravenously at a dose ranging from about 0.1 mg/ml/kg to 20 mg/ml/kgor more preferably from about 1 mg/ml/kg to 10 mg/ml/kg. In anotherembodiment, the S-nitrosylating agent is ethyl nitrite that isadministered by atomizer, diluted in ethanol at a concentration of about1-100 part per trillion, or 10 mg delivered orally three times per dayorally. In an alternative embodiment nitric oxide is administered(especially for newborns) continuously by inhalation in air/oxygen indoses of 100-500 parts per trillion, or qid doses of 10 ppm, to increasered blood cell SNO stores.

[0025] When administered orally, the compounds of the present inventioncan be administered as a liquid solution, powder, tablet, capsule orlozenge. The nitrosylating agents can be used in combination with one ormore conventional pharmaceutical additives or excipients used in thepreparation of tablets, capsules, lozenges and other orallyadministrable forms. When administered as an intravenous solution, thenitrosylating agents can be admixed with conventional IV solutions.

[0026] In accordance with one embodiment pharmacological agents areprovided to treat apnea, hypoventilation, impending respiratory failureand hyperventilation. In particular, in one embodiment a compositioncomprising a low molecular weight reduced thiol is administered to anindividual to treat a disorder of control of breathing. Moreparticularly, the composition comprises a reduced glutathione,N-acetylcysteine, cysteinylglycine, and L-cysteine or other agents thatfunction to increase delivery of S-nitrosothiols to neural respiratorycontrol centers. The composition is formulated with pharmaceuticallyacceptable carriers, diluents and excipients for administration via anoral, parenteral or inhalation route. In accordance with one embodiment,compositions comprising a reduced thiol are used to treat centralhypoventilation associated with sleep, whether congenital (as incongential central hypoventilation syndrome) or acquired (such as isseen in sleep apnea syndromes, including obstructive sleep apnea). WhenN-acetylcysteine (generic) is selected as the S-nitrosylating agent thepreferred route of administration is orally in doses of 10 mg threetimes per day by mouth (or in the range of 0.5-1.0 mg/kg qid) for apnea.

[0027] In another embodiment of the present invention a method isprovided for treating impaired respiratory drive associated withemphysema, chronic bronchitis, cystic fibrosis, α-1 antitrypsindeficiency and other causes of chronic obstructive pulmonary disease.The method comprises the step of administering a reduced thiolcomposition of the present invention. Furthermore the compositions ofthe present invention can be used to permit oxygen therapy in thesepatients, whose disease is characterized by a blunted response to carbondioxide and a dependence on hypoxic ventilatory drive (which is in turnis blunted with supplemental oxygen therapy). The nitrosylating agentsof the present invention can overcome such a blunted respiratory driveby directly stimulating the neural respiratory control centers locatedin the brain stem and the carotid body.

[0028] In another embodiment the reduced thiol compositions of thepresent invention are used to stimulate respiration in severe, hypoxicrespiratory distress associated with impending respiratory failure. Thegoal of this therapy is to allow an increase in minute ventilation boththrough improved S-nitrosothiol delivery to the brain stem, and improvedoxygen delivery to the periphery (to obviate the need for mechanicalrespiratory support). In accordance with one embodiment a method oftreating blunted respiratory drive comprises the step of administering,preferably by intravenous injection, a composition comprising anitrosylating agent selected from the group consisting of ethyl nitrite,glutathione, S-nitrosoglutathione, S-nitroso-L-cysteinyl glycine,S-nitrosocysteine, N-acetyl cysteine, S-nitroso-N-acetyl cysteine andnitric oxide.

[0029] In another embodiment of the present invention a method isprovided for treating central apnea caused by brain stem lesions(including tumors, radiation injury, trauma, chronic scarring, bleedingsuch as aneurysms or arterial venous malformation, tethering of thespinal cord from Arnold Chiari malformation and/or brain stemcompression from hydrocephalous, neonatal anemia and brain stemimmaturity associated with the newborn and/or premature newborn state).The method comprises the steps of administering to a patient in needthereof, a composition comprising an S-nitrosylating agent, includingethyl nitrite, S-nitrosoglutathione, S-nitrosocysteinyl glycine,S-nitrosocysteine, nitroglycerine, nitroprusside, nitric oxide,S-nitrosoanastylcysteine, S-nitrosohemoglobin and S-nitrosoalbumin. Thecomposition can be administered orally, parenterally or by inhalation.

[0030] The mechanism by which these nitrosylating agents act is toincrease the concentration of circulating S-nitrosothiols and/or theS-nitrosylation of endogenous thiol groups in erythrocytes to have a netaffect of increasing S-nitrosylation of neural respiratory controltargets. In one embodiment compositions comprising S-nitrosylatingagents are used to treat central hypoventilation associated with sleep,whether congenital (as in congential central hypoventilation syndrome)or acquired (such as is seen in sleep apnea syndromes, includingobstructive sleep apnea). In another embodiment the compositionscomprising S-nitrosylating agents are used to treat impaired respiratorydrive associated with emphysema, chronic bronchitis, cystic fibrosis,α-1 antitrypsin deficiency and other causes of chronic obstructivepulmonary disease. Furthermore, such compositions can be used to permitoxygen therapy in these patients, whose disease is characterized by ablunted response to carbon dioxide and a dependence on hypoxicventilatory drive (which is in turn blunted with supplemental oxygentherapy), and thus stimulate respiration in severe, hypoxic respiratorydistress associated with impending respiratory failure. The goal of thistherapy is to allow an increase in minute ventilation both throughimproved S-nitrosothiol delivery to the brain stem, and improved oxygendelivery to the periphery (to obviate the need for mechanicalrespiratory support).

[0031] The present invention is also directed to any composition thateffects an increase in S-nitrosocysteinyl glycine levels in vivo eitherdirectly or indirectly. For example a therapeutic composition mayinclude one or more agents that result in an increased cleavage ofS-nitrosoglutathione to active S-nitrosocysteinyl glycine (see FIG. 2).Such agents include stimulants of γ-glutamyl transpeptidase expressionand/or γ-glutamyl transpeptidase activity. For example such stimulantsinclude retinoic acid and other retinols and stimulants of retinoic acidreceptors, follicle stimulating hormone, ethoxyquin and other stimulantsof GGT promoter III or inhibitions of GGT gene negative regulatorysites, glycine-glycine in conjunction with glutathione, prostaglandin E2with diethyInitrosamine, and stimulants of the antioxidant responseelement promoter. Compositions comprising such stimulants of GGTactivity can be used in a method of treating central apnea caused bybrain stem lesions (including tumors, radiation injury, trauma, chronicscarring, bleeding such as aneurysms or arterial venous malformation,tethering of the spinal cord from Arnold Chiari malformation and/orbrain stem compression from hydrocephalous, neonatal anemia and brainstem immaturity associated with the newborn and/or premature newbornstate).

[0032] In another embodiment, a composition comprising a stimulant ofGGT is used to treat central hypoventilation associated with sleep,whether congenital (as in congential central hypoventilation syndrome)or acquired (such as is seen in sleep apnea syndromes, includingobstructive sleep apnea). Such a composition can also be used to treatimpaired respiratory drive associated with emphysema, chronicbronchitis, cystic fibrosis, a-1 antitrypsin deficiency and other causesof chronic obstructive pulmonary disease. Further, these agents can beused to permit oxygen therapy in these patients, whose disease ischaracterized by a blunted response to carbon dioxide and a dependenceon hypoxic ventilatory drive (which is in turn blunted with supplementaloxygen therapy) and stimulate respiration in severe, hypoxic respiratorydistress associated with impending respiratory failure. The goal of thistherapy would be to allow an increase in minute ventilation both throughimproved S-nitrosothiol delivery to the brain stem, and improved oxygendelivery to the periphery (to obviate the need for mechanicalrespiratory support).

[0033] Alternatively, the systemic use of an inhibitor of GGT such asacivicin can be used to treat respiratory alkalosis associated withpsychiatric hyperventilation or salicylate toxicity. The methodcomprises administering a composition comprising an inhibitor of GGT toa patient suffering from psychiatric hyperventilation or salicylatetoxicity. In addition, inhibition of GGT can also be used acutely totreat hyperventilation associated with psychiatric disorders and thetoxicity of certain medications such as aspirin. Preferred routes ofadministration include oral, parenteral or inhalation.

[0034] The present invention also encompasses a pack or kit comprising aa nitrosylating agent selected from the group consisting of ethylnitrite, glutathione, S-nitrosoglutathione, S-nitroso-L-cysteinylglycine, S-nitrosocysteine, N-acetyl cysteine, S-nitroso-N-acetylcysteine and nitric oxide, for treating disordered control of breathing.The kits of the present invention may further comprise reagents fordetecting and monitoring the in vivo concentration of S-nitrosothiols aswell as syringes and other materials for administering the nitrosylatingagents of the present invention. The nitrosylating agents of the kit canbe packaged in a variety of containers, e.g., vials, tubes, microtiterwell plates, bottles, and the like. Other reagents can be included inseparate containers and provided with the kit; e.g., positive controlsamples, negative control samples, buffers, cell culture media, etc.Preferably, the kits will also include instructions for use.

Example 1

[0035] SNOs Stimulate Respiratory Centers of the NTS to Increase V_(E).

[0036] To test the hypothesis that SNOs stimulate respiratory centers ofthe NTS to increase V_(E), the ability of endogenous SNOs to increaseV_(E) in freely behaving, conscious rats using whole-bodyplethysmography was examined. CSNO, GSNO and CGSNO (1 nmol each) causedequivalent increases in V_(E), whereas D-CSNO had no effect (see FIG. 1;dose threshold for L-SNOs is 0.1 pmol). The exogenous NO donor,S-nitroso-N-acetyl-L-penicillamine, had similar but reduced effects (notshown). The L- and D-isomers of CSNO decayed at identical rates in ratbrainstem homogenates (26% min-1 mg -1 protein each; P=not significant(NS)). Furthermore, neither excess 8-bromocyclic GMP nor glutathione hadany effect on VE (n=3;P=NS).

[0037] Next, a low-mass fraction (less than a relative molecular mass of10,000 (Mr 10K)) derived from deoxygenated blood was studied todetermine whether the fraction would similarly increase V_(E). Thisfraction reproduced precisely the effect of GSNO, L-CSNO and CGSNO,whereas the fraction from oxygenated blood was without effect. Asexpected, ultraviolet photolysis of the deoxygenated, blood-derivedfraction (which causes homolytic cleavage of the SNO bond and liberationof free NO) completely eliminated its effect on V_(E) (n=3). Theseobservations suggest that SNOs arising during blood deoxygenation cansignal an increase in V_(E).

[0038] The normal physiological response to hypoxia was then studied inrelation to the SNO effect on V_(E). Exposure to a 10% oxygenenvironment resulted in an increase in V_(E) identical to that producedby L-isomers of SNOs administered to the NTS (FIG. 1A). The decaycharacteristics for the recovery of V_(E) after injection of GSNO wereidentical to those for recovery from hypoxia (FIGS. 1A & 1B). Thisrecovery is characterized by a return of V_(E) to baseline overapproximately 3 min after return to normoxia. Taken together, theseobservations demonstrate that SNOs duplicate the physiological responseof exposure to, and recovery from, hypoxia.

[0039] Endothelial transport and targeted neuronal biological activityof GSNO may depend, under certain circumstances, on biochemicalmodifications such as cleavage by GGT to form CGSNO. More particularly,NTS pre-treatment with the GGT inhibitor, acivicin, was found toattenuated GSNO-mediated increases in V_(E), and that CGSNO overcamethis inhibition (FIG. 2). Moreover, normal ventilatory offset (that is,the gradual return to baseline of V_(E) after recovery from hypoxia thatprevents apnea) was inhibited by acivicin. This suggests that GSNO is acritical precursor of CGSNO (and perhaps dipeptidase derived L-CSNO) indetermining hypoxic ventilatory responses in vivo (FIG. 2). As expected,deoxygenated blood-derived CGSNO was found to be less stable than GSNO,consistent with a pathway by which GSNO is activated locally by GGT inneuronal tissue. Of note, this effect of GGT may distinguish respiratorystimuli to the NTS (which are reproduced by GSNO and are GGT dependent)from haemodynamic effects of SNOs, which although stereoselective arenot reproduced by GSNO. This distinction may have pharmacologicalimplications.

[0040] Finally, GGT was demonstrated to be required for the normalventilatory response to hypoxia using a mouse deficient in GGT.Homozygous deficient mice had profound attenuation of hypoxicventilatory recovery (FIG. 2C). These results show that endogenous SNOsact stereoselectively at the level of the NTS to produce the normalventilatory response seen during hypoxia. This fits well with theproposal that SNOs may serve as signaling molecules between endothelialcells and central and peripheral neurons, as well as recent observationsthat (1) hemoglobin deoxygenation is associated with an increase in SNOformation and biological activity; and (2) neuronal tissues contain highlevels of SNOs afferents from peripheral respiratory chemoreceptorsproject to areas of the NTS rich in NOS, which may produce SNOs; NOSexpression by NTS is increased after chronic hypoxia; and hypoxicventilatory responses are attenuated both by NOS inhibition and inendothelial NOS knockout mice.

[0041] In addition, a compound derived from deoxygenated, but notoxygenated, blood reproduces the ventilatory effect of hypoxia. Thisbiological activity is physiologically identical to both exogenous SNOsand hypoxia itself, and was identified as GSNO by mass spectrometry.Both this SNO activity and hypoxia require GGT for normal signaling.Additional responses to hypoxia attributed to the transfer of nitrogenoxides from deoxygenated blood include vasodilation to maintain oxygendelivery and increases in endothelial gene expression mediated byhypoxia-inducible factor-1. S-nitrosothiol signaling is believed to beof central importance in the normal response to hypoxia, and it isanticipated that this pathway will provide targets for the developmentof new treatments for apnea

[0042] Methods

[0043] Measurement of V_(E)

[0044] A dual cannula (22G; Plastics One) was implanted close to thenucleus of the solitary tract according to standard stereotaxiccoordinates (−14.0 mm bregma, 0.5 mm off midline, 7.0 mm depth; seePaxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates(Academic, New York, 1986), FIG. 74) through a hole drilled into theoccipital skull of male Sprague-Dawley rats (approximately 250 g;pentobarbital anaesthesia). Placement was confirmed histologically afterprotocol completion. After recovery for 48 h (return to normal feedingand sleep/waking patterns), breath-by-breath ventilation was measuredusing the barometric method previously described (Gozal et al., J. Appl.Physiol. 81, 2068-2077 (1996)) after simultaneous, bilateral 0.1-ulinjections in the freely behaving animal.

[0045] Plasma Preparation

[0046] Oxygenated blood drawn in an airtight syringe with GSH and EDTA(final concentrations 400 μM) by left ventricular puncture (rat) orperipherally (human) was maintained in 21% oxygen. Venous blood wasreacted identically and was transferred to a hypoxia chamber underargon. Oxygen tension was measured electrochemically (Chirion). Samplesunderwent centrifugation (3,000 g for 5 min) followed by ultrafiltration(10K; Millipore), separation and selective ultraviolet photolysis(Jelight PS-3000-30; Laguna-Hills).

[0047] Preparation of and assay for S-nitrosothiols SNOs were preparedby acid nitrosation, titrated to pH 7.4 and maintained in EDTA in thedark at −80° C. until use to prevent decomposition. SNOs were detectedby reduction/chemiluminescence as previously described (Fang et al.,Biochem. Biophys. Res. Commun. 252, 535-540 (1998)). Briefly, sampleswere injected into 100 μM CuCl, 1 mM cysteine (pH 6; 50 8C) and purgedwith helium (grade 5; BOC Gases). Evolved NO was measured bychemiluminescence.

[0048] Mass Spectrometry

[0049] Samples eluted isocratically (90% of 0.1% formic acid, 10%methanol, 1.5 ml min⁻¹) over a Waters Symmetry C18 column (7.8×150 mm)were collected, lyophilized and reconstituted. Purified samples wereinjected onto a Waters C18 microbore column (1.0×150 mm) and analyzed byelectrospray ionization mass spectrometry using a Finnigan LCQ Duosystem. GSNO cations were monitored by selective ion monitoring at amass to charge ratio (m/z) of 336.9. For mass spectrometry and massspectrometry fragmentation experiments, GSNO cations were dissociated inthe ion trap, and the fragments were monitored within a m/z range of90±350.

1. A method of treating disordered control of breathing, said methodcomprising the step of administering an S-nitrosylating agent selectedfrom the group consisting of ethyl nitrite, glutathione,S-nitrosoglutathione, S-nitroso-L-cysteinyl glycine, S-nitrosocysteine,N-acetyl cysteine, S-nitroso-N-acetyl cysteine and nitric oxide.
 2. Themethod of claim 1 wherein the S-nitrosylating agent is selected from thegroup consisting of glutathione, S-nitrosoglutathione, N-acetyl cysteineand S-nitroso-N-acetyl cysteine and the composition is administeredintravenously.
 3. The method of claim 1 wherein the S-nitrosylatingagent is selected from the group consisting of N-acetyl cysteine,S-nitroso-N-acetyl cysteine, and S-nitrosoglutathione and thecomposition is administered by inhalation.
 4. The method of claim 1wherein the S-nitrosylating agent is selected from the group consistingof N-acetyl cysteine, S-nitroso-N-acetyl cysteine, andS-nitrosoglutathione, and the composition is administered orally.
 5. Akit for treating disordered control of breathing, said kit comprising anS-nitrosylating agent selected from the group consisting of ethylnitrite, glutathione, S-nitrosoglutathione, S-nitroso-L-cysteinylglycine, S-nitrosocysteine, N-acetyl cysteine, S-nitroso-N-acetylcysteine and nitric oxide and reagents for detecting and monitoring thein vivo concentration of S-nitrosothiols.
 6. The kit of claim 5 furthercomprising an agent that results in an increased cleavage ofS-nitrosoglutathione to active S-nitrosocysteinyl glycine.
 7. The kit ofclaim 6 wherein said agent has γ-glutamyl transpeptidase activity.
 8. Amethod of increasing minute ventilation (V_(E)) at the level of thebrainstem respiratory control centers in the nucleus tractus solitariusof an individual, said method comprising the step of administering tosaid individual a composition comprising an S-nitrosylating agent. 9.The method of claim 8 wherein the S-nitrosylating agent is selected fromthe group consisting of ethyl nitrite, glutathione,S-nitrosoglutathione, S-nitroso-L-cysteinyl glycine, S-nitrosocysteine,N-acetyl cysteine, S-nitroso-N-acetyl cysteine and nitric oxide.
 10. Themethod of claim 9 wherein the S-nitrosylating agent is selected from thegroup consisting of N-acetyl cysteine, S-nitroso-N-acetyl cysteine, andS-nitrosoglutathione and the composition is administered by inhalation.11. The method of claim 9 wherein the S-nitrosylating agent is selectedfrom the group consisting of N-acetyl cysteine, S-nitroso-N-acetylcysteine, and S-nitrosoglutathione, and the composition is administeredorally.